A toxin from Pasteurella multocida type D causes acute hepatic necrosis in pigs.

Abstract

Toxins of Pusteurellu multocidu type D, a causal bacterium of porcine atrophic rhinitis, chiefly affect developing cartilage and bone. When sprayed onto nasal mucosae or injected subcutaneously, they cause lesions of nasal turbinates within 48 h r . * ~ ~ Systemic effects of pasteurella toxins have not been clearly shown but are suggested by two experimental studies in pigs: (1) toxin given by aerosol caused hepatocyte damage in six of ten pigs-lesions varied from focal vacuolar degeneration to diffuse necrosis and hemorrhage;2 (2) toxin given intraperitoneally caused liver lesions which were defined only as “degenerative change.”8 Since clinical studies on atrophic rhinitis hint that the liver may be affected in natural disease, we wished to know if the pig developed liver necrosis when given toxin experimentally. Six piglets (specific pathogen-free, 3 days old) were given 0.1 pg (0.1 ml) toxin subcutaneously; two controls (pigs 1, 2) were given diluent subcutaneously. Two pigs were to be necropsied at 2 hr (pigs 3, 4), 24 hr (pigs 5, 6), and 48 hr (pigs 7, 8) after toxin (Table 1). Toxin, purified from noncapsulated Pusteurellu multocidu strain P-4533,4J was suspended in buffer (0.05 M Tris, 0.1 M NaC1,0.002 M EDTA, 0.5% bovine serum albumin, pH 8.3) at a protein concentration of 1 pg/ml; the lethal dose for swine is between 0.1 and 0.8 pg/kg given intradermally.“ From all pigs, slices of liver, kidney, lung, adrenal, stomach, jejunum, thymus, spleen, mesenteric lymph node, and rib were fixed in 10% formalin and processed by standard histologic techniques. After embedment in paraffin blocks, sections (6 pm) were cut and stained with hematoxylin and eosin (HE), Giemsa, oil red 0, and alcian blue-periodic acid-Schiff (with and without diastase) techniques. Livers of normal control piglets had no pathologic changes; hepatocytes contained large amounts of glycogen but little lipid. Livers sampled at 2 hr after toxin (Fig. 1) resembled controls except for three changes, all of which were slight: (1) loss of glycogen and increased density of cytoplasm of hepatocytes surrounding central veins; (2) dilation of the hepatic venous system; and (3) infiltrates of small numbers of neutrophilic leukocytes in surrounding sinusoids. Pathologic changes in livers at 24 hr after toxin were striking and severe (Fig. 2). They included: (1) marked intrahepatic edema; ( 2 ) diffuse swelling and enlargement of Kupffer cells; (3) diffuse endothelial swelling and necrosis with dilation of sinusoids; (4) diffise degeneration of hepatocytes; and (5) random foci of necrosis, which were especially prominent at the surface of the liver. Foci of necrosis were characterized by detachment of necrotic hepatocytes, insudation of plasma proteins, and microcavitation (Fig. 3). Pig 7 died 33 hr after toxin, and pig 8 was killed 36 hr after toxin; lesions did not differ from those in 24-hr samples. Degenerate hepatocytes throughout the livers of piglets sampled at 24 and 36 hr after toxin had marked depletion of glycogen, vacuolar degeneration, and increased basophilia of cytoplasm. Small vacuoles in the cytoplasm of hepatocytes were lucent and did not stain for fat or glycoproteins. In contrast, larger vacuoles contained inspissated protein which stained with the periodic acid-Schiff (PAS) technique. Some appeared as hyalin lakes that filled the cytoplasm (Fig. 4). Endothelial degeneration and necrosis in livers at 24 hr after toxin was diffuse but was most severe in terminal sinusoids and central veins. Neutrophils were prominent in these areas. Perivascular eosinophilic precipitates of protein were around most central veins. Lesions in other organs were more variable than the hepatic lesions. No significant changes were seen 2 hr after toxin. In 24and 36-hr samples, in addition to marked generalized hyperemia, histologic lesions included: (1) small intestine: necrosis of capillaries of the tips of villi with many degenerate rounded enterocytes free in the gut lumen close to the villous tips (Fig. 5); (2) kidney: acute cell swelling and necrosis of segments of the proximal convoluted tubules in developing nephrons (Fig. 6), especially adjacent to developing glomeruli at the surface of the kidney; (3) lungs: slight edema of the alveolar wall with dilation of capillaries, neutrophilic leukocytes within and external to blood vessels, dilatation of lymphatics, and endothelial damage; (4) bone: necrosis of capillaries and fibrin deposition at the junction of cartilage and bone at the growth plate; and ( 5 ) adrenal: focal necrosis in the cortex of two of four glands. No significant lesions were seen in pancreas, thymus, lymph node, or spleen. Studies on blood collected just before necropsy did not reveal marked changes in leukocytes or plasma proteins (Table 1). Complement levels were not significantly reduced. There were consistent elevations in serum sorbitol dehydrogenase associated with liver injury. These studies show that pasteurella toxin causes hepatic’ necrosis associated with widespread vascular injury and vacuolar degeneration of hepatocytes. The mechanism of toxin action on hepatocytes appears to be associated with, but